Neutrophils are professional migrators within our body, moving from the bloodstream through dense forests of extracellular matrix to reach sites of infection and initiate an immune response. While we have long appreciated the role of biochemical signals in neutrophil recruitment, increasing attention is being given to the role of physical signals during this process. For example, membrane tension and membrane shape changes arise as neutrophils navigate complex 3D environments in vivo, which can impact the path cells take through tissue. However, how this information is relayed back to the cytoskeleton has remained largely unexplored. In this thesis we probe the link between biochemical and physical inputs to better understand how neutrophils integrate environmental signals to ensure robust directed migration.

In Chapter 2 we establish how intrinsic protein self-organization and external membrane curvature work together to pattern the actin nucleation promoting factor WAVE. Using super-resolution microscopy, we find that WAVE forms rings at the saddle-shaped necks of membrane invaginations in the absence of actin polymer. Dual inputs from WAVE’s propensity to oligomerize and curvature sensitivity explain this enrichment pattern and inform our understanding of WAVE propagation in the presence of actin. Specifically, we find that WAVE localizes to the saddle-shaped lagging regions of the leading edge to support a uniformly advancing cell front. Elucidation of the mechanisms underpinning WAVE organization has provided initial insights into the integration of internal biochemical signals with external cell shape in organizing the actin cytoskeleton.

In Chapter 3 we continue on this theme and highlight how WASP, another actin nucleation promoting factor, uses a modification of these behaviors to link cell shape and cell polarity during neutrophil migration. Unlike WAVE, WASP persistently enriches to substrate-induced invaginations in the presence of actin. Additionally, WASP assembles into focal structures rather than linear structures. We find that these properties allow WASP to link sites of local membrane deformation to the cytoskeleton by promoting actin polymerization and aligned migration in textured environments. Strikingly, this property requires concomitant inputs from cell shape and cell polarity. WASP only engages with curved membranes at the cell front, which supports forward advance. We show that WASP is essential for integration of substrate features with directed migration, which could have implications for migration through collagen meshworks in vivo.

Finally, Chapter 4 presents unpublished work exploring the molecular mechanisms of cell polarity through GTPase regulation by Rho GEFs and GAPs using HEK293T cells as a model system. Using CRISPR we endogenously label highly expressed GEFs and GAPs. We describe a system for polarizing HEK293T cells and report candidate GEFs and GAPs that enrich to induced cell protrusions. Finally, we supply a protocol for phenotyping the migration of HEK293T GEF or GAP knockout cell lines and provide a positive control in the form of Rac1 knockout. Ultimately, GEFs and GAPs exhibiting loss of function phenotypes would be followed up in the consitituively polarized, highly motile neutrophil cell line HL-60.

This thesis presents work toward understanding the biophysical mechanisms that control polarized cell movement.  Many cells move in response to external cues, and this directed movement occurs during development, axon guidance, wound healing, tumor progression, and the immune response.  Cells must interpret external signals through signal transduction networks and engage actin polymerization machinery to generate mechanical force for membrane protrusion at the leading edge of the cell.  Our work focuses specifically on the interface between the cell's signaling guidance system and actin nucleation machinery.

In the first project, we analyzed the protein dynamics of a major cytoskeleton regulator called the WAVE complex in a human neutrophil-like cell line in response to gradient and uniform agonist stimulations (Chapter 3). We demonstrated that biased generation and selection of WAVE-complex activity drives the development of morphological polarity and we discovered that actin polymer sculpts the localization of the WAVE-complex.

In the second project, we developed a biochemical system to study the WAVE complex, discovered a GTPase exchange factor likely involved in WAVE-complex activation, searched for other WAVE-complex binding proteins through immunoprecipitation, and examined cell behavior in response to temporal changes in agonist (Chapter 4).

In the third project, we studied the single-molecule behavior of cytoskeletal proteins in XTC cells using epifluorescence and TIRF microscopy. We showed that the WAVE complex intercalates into the actin network during retrograde grade flow. Additionally, we observed lateral diffusion of both the WAVE complex and the Arp2/3 complex and captured the transition of the Arp2/3 complex from diffusion to network incorporation.

The kinetics and intensity of stimulation are thought to affect T cell signaling at multiple levels. For example, at the level of the receptor, ligand binding kinetics have been suggested to discriminate agonistic from non-agonist pMHCs. Downstream of the receptor, small changes in the intensity of stimulation can sharpen into a large all-or-nothing activation of Erk. However, precisely tuning the kinetics and intensity of stimulation and measuring the effects has remained challenging, limited in part by a lack of appropriate tools. Here we report the use of a LOV2-based optogenetic system to control the kinetics and intensity stimulation through a chimeric antigen receptor. Signaling through the receptor is initiated by the binding of LOV2 to the extracellular binding domain. Varying the intensity of blue light tunes the ligand binding kinetics from sub-second to tens of seconds. Other biophysical aspects of the interaction are not changed, allowing us to cleanly measure the effects ligand binding kinetics have on receptor signaling. Downstream of the receptor, titrating the intensity of blue light titrates the strength of the signal. By using several fluorescent live cell readouts of proximal signaling, we can measure single cell “dose response curves,” seeing if different portions of the signaling pathway respond in a linear, switch-like, adaptive or integrated manner to upstream signals. Optogenetic control of receptor signaling allows us to study T cell signaling with greater control than ever before, allowing us to directly ask how cells respond to changes in the kinetics and intensity of stimulation.

This thesis presents work toward understanding how chick neural crest cells polarize their protrusions to achieve directed motility. There are multiple strategies that cells can use to generate directed migration. They can read gradients of guidance cues spatially across their surface or compare the concentration of agonist in time. Cells can bias the generation of protrusions or their lifetime. Determining the logical framework that cells use for migration is often challenging as it is not readily deduced from knockout phenotypes.

Here we use 3D two-photon imaging of chick branchial arch 2 directed neural crest cells to probe how these mechanisms contribute to directed movement (Chapter two). We find that cells throughout the stream are morphologically polarized along the direction of overall stream movement and that there is a progressive sharpening of the morphological polarity program. Neural crest cells have weak spatial biases in filopodia generation and lifetime. Local bursts of filopodial generation precede the generation of larger protrusions. These larger protrusions are more spatially biased than the filopodia, and the subset of protrusions that power motility are the most polarized of all. Orientation rather than position is the best correlate of the protrusions that are selected for cell movement. This progressive polarity refinement strategy may enable neural crest cells to efficiently explore their environment and migrate accurately in the face of noisy guidance cues.

Neural crest cell migration occurs in a 3D environment. However, published protocols for culturing migratory neural crest cells and performing chemotaxis assays have all made use of 2D culture. We sought to determine whether neural crest cell morphology in 2D culture systems resembled that in vivo and if not to explore whether 3D culture techniques could improve neural crest cell behavior (Chapter 3).

This thesis presents work toward understanding the spatial organization of key molecules during cell morphogenesis and migration. Cell migration is essential for many processes including developmental morphogenesis, axon guidance, and immune responses. Chemotaxis, or directed migration guided by chemical cues, requires the spatial and temporal coordination of a multitude of molecules that pattern the force-generating actin cytoskeleton to build plasma membrane protrusions and power cell motility. This work focuses on identifying novel chemotaxis effectors, dissecting their molecular signaling logic, and exploring how key molecules spatially organize to enable the large-scale, self-organization of cell shape and movement.

In the first project, we identified and characterized a novel signaling effector of neutrophil chemotaxis (Chapter 2). From a mass spectrometry pulldown screen, we identified Homer3 as a Gαi2 interacting protein. With biochemical and cell biology techniques, we report that Homer3 is necessary for efficient chemotaxis by regulating the polarized spatial organization, rather than the magnitude and kinetics, of key signaling molecules. Overall, our work characterized how Homer3 functions as a scaffold to spatially organize polarity signaling and actin assembly.

In the second project, we studied the spatial organization of the WAVE complex, which is a key effector of cell shape and migration across eukaryotes (Chapter 3). Using quantitative, live-cell super-resolution microscopy, we discovered how the WAVE complex spatially assembles into nanometer scale ring structures at sites of saddle membrane curvature in the absence of actin polymerization. This geometric association for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes.

In the third project, I describe my pilot work using nanotopography to physically manipulate cell geometry to assay curvature sensation (Chapter 4). The interdisciplinary nature of this experiment, which spans nano-engineering, cell biology, and high-resolution microscopy, highlights a combination of expertise that will undoubtedly unveil exciting insights.

During gastrulation, endodermal cells actively migrate to the interior of the embryo, but the signals that initiate and coordinate this migration are poorly understood. By transplanting ectopically-induced endodermal cells far from the normal location of endoderm specification, we identified the inputs that drive internalization without the confounding influences of fate specification and global morphogenic movements. We find that Nodal signaling triggers an autocrine circuit for initiating endodermal internalization. Activation of the Nodal receptor directs endodermal specification through sox32 and also induces expression of more Nodal ligands. These ligands act in an autocrine fashion to initiate endodermal cell sorting. Our work defines an “AND” gate consisting of sox32-dependent endodermal specification and Nodal ligand reception controlling endodermal cell sorting to the inner layer of the embryo at the onset of gastrulation.

Neutrophils are one of the body's first lines of defense against pathogens. These cells can sense the molecular signatures of injury and infection and polarize their cytoskeletons in a single direction to move toward the sources of these cues. Previous work has detailed how these cells polarize toward signals, but it is just as important to understand how they adjust their direction over time. Neuroscientists have used virtual reality systems to understand how organisms process and respond to environmental information; we used optogenetics to perform comparable experiments in neutrophil-like HL-60 cells by controlling a key polarity signal in cells as they migrated. For example, by presenting cells with two polarity sites in various configurations, we tested which local features predicted the cell's preference for one stimulus compared to another. Using assays like this, we could tease apart which spatial and temporal characteristics of input signals controlled cell directionality. We found that the fronts of cells respond to the local rate-of-change in input signals and that the backs of cells are generally unresponsive. Migrating cells adjust direction based on where within the front they are experiencing the fastest increase in signal.